Growth inhibition and apoptosis induction of Scutellaria luteo-coerulea Bornm. & Sint. on leukemia cancer cell lines K562 and HL-60

Objective: Scutellaria (Lamiaceae) has been implicated for medicinal purposes both in modern and traditional medicine. Some species of the genus Scutellaria has extensively been studied for anticancer activity. Scutellaria luteo-coerulea (S. luteo-coerulea) is one of the Iranian species of the genus Scutellaria. Materials and Methods: In the present study, cytotoxic and apoptogenic properties of CH2Cl2, EtOAc, n-BuOH, and H2O fractions of S. luteo-coerulea were investigated on K562. Moreover, HL-60. DNA fragmentation in apoptotic cells were determined by propidium iodide (PI) staining (sub-G1 peak). Results: Scutellaria luteo-coerulea inhibited the growth of malignant cells in a dose-dependent manner. Among solvent fractions of S. luteo-coerulea, the CH2Cl2 fraction was found to be the most cytotoxic one among others. Sub-G1 peak in flow cytometry histogram of treated cells suggested the induction of apoptosis in S. luteo-coerulea. Conclusion: Scutellaria luteo-coerulea could be a novel candidate for further analytical elucidation in respect to fine major components responsible for the cytotoxic effect of the plant also clinical evaluations.


Introduction
The genus Scutellaria (Lamiaceae) with about 360 different species, is one of the popular herbs which has been extensively investigated in biological researches. Some plants of the genus Scutellaria have been used in traditional and folk medicine. Since 1889, phenolic (flavonoids, phenylethanoid glycosides) and terpene compounds (iridoid glycosides, diterpenes, and triterpenoids) have been identified as two main groups of constituents of Scutellaria.
Baicalin, baicalein, wogonin, and neobaicalein are among the main compounds of flavonoids possess anti-cancer, anti-microbial, anti-inflammatory, and anticonvulsant effects (Shang et al., 2010). Baicalein and baicalin flavonoids were shown to have protective properties against reactive oxygen species (ROS)induced tissue damage and antimicrobial activity. The inhibition of HIV-1 reverse transcriptase has also been reported for baicalein (Mamadalieva et al., 2011). Wogonin, baicalein and baicalin as the major constituents of S. baicalensis have been shown cytotoxic activity to various human tumor cell lines with minor toxicity to normal cells (Li-Weber, 2008). Neobaicalein an active ingredient of the S. litwinowii exerts cytotoxic and pro-apoptotic effects in human cervical cancer, HeLa cell line (Tayarani-Najarani et al., 2012).
Scutellaria colebrookiana and S. violacea showed cytotoxic and antioxidant potential which may attributed to the presence of baicalein (Salini et al., 2013). Scutellaria baicalensis and S. rivularis have been revealed inhibitory effect on HL-60 cells which is related to flavonoids in the plant (Sonoda et al., 2004). Baikal skullcap (S. baicalensis) and S. barbata possess anti-proliferative and apoptosis-inducing activities (Yu et al., 2007;Kim et al., 2007). Scutellaria radix has an antiproliferative effect on myeloma cells and baicalein may be responsible for the inhibitory activity of Scutellaria radix by blocking IkB-degradation (Ma et al., 2005) , 2010). Scutellaria baicalensis has also had strong inhibitory effect on cell growth in prostate and breast cancer cells (PC-3, LNCaP, and MCF-7) which may partly be due to its ability to inhibit COX-2 activity (Ye et al., 2002).
In the course of cytotoxic activity stated for several Scutellaria species, the present paper describes the antiproliferative and apoptogenic activity of S. luteo-coerulea, one of the Iranian species of Scutellaria. There is not any reported literature on S. luteo-coerulea. In order to give insight to potential cytotoxic activity of the S. luteo-coerulea, CH 2 Cl 2, and EtOAc, n-BuOH and H 2 O fractions of the plant have been evaluated on K562 and HL-60 cells. The role of apoptosis in this cytotoxicity was also assessed.

Plant materials
Roots of S. luteo-coerulea were collected from Tandooreh

Cell cultures
The human leukemia cancer cells (K562 and HL-60) were obtained from Pasteure institute (Tehran, Iran) and maintained in RPMI-1640 medium supplemented with 10% fetal bovine serum, 100 U/mL penicillin, and 100 µg/mL streptomycin at 37 ºC in a humidified atmosphere of 95% air and 5% CO 2 . The stock solution of each compound was prepared at 50 mg/mL in dimethylsulfoxide and kept at -20 °C.
For MTS and apoptosis assay, 10 4 cells/well and 10 5 cells/well were seeded to 96-well and 24-well plates, respectively.
Each assay was compared with a untreated control received the equal volume of DMSO.

Cell viability
The -2H-tetrazolium inner salt (MTS) by mitochondrial dehydrogenase in live cells produce the colored, water-soluble formazan that absorbs at 490 nm (Malich et al., 1997

PI staining
PI stained cells were evaluated by flow cytometry to detect the so-called sub-G1 peak in apoptotic cells (Nicoletti et al., 1991). Briefly, 10 5 K562 and HL-60 cells were seeded in each well of a 24-well plate and treated with CH 2 Cl 2 fraction of S. luteo-coerulea different concentrations (0-125 µg/mL) for 48 h. Floating and adherent cells were then harvested and incubated with 50 μg/mL PI in 0.1% sodium citrate plus 0.1% Triton X-100. Small fragments of DNA in apoptotic cells were eluted following incubation in a hypotonic phosphate-citrate buffer and stained with PI before flow cytometric analysis using a FACScan flow cytometer (Becton Dickinson) was done. 10 4 events were acquired with FACS.

Statistical analysis
One way analysis of variance (ANOVA) and Bonferroni's post hoc were used for data analysis. All results were expressed as mean±SEM and p values below 0.05 were considered statistically significant.

Inhibition of cell viability
Inhibition of cell viability caused by fractions extract of S. luteo-coerulea was examined using MTS assay.
In order to compare the cytotoxicity of fractions extract of S. luteo-coerulea the K562 and HL-60 cells were incubated with different concentrations for 48 h. The results showed these compounds decreased cell viability of cells in a concentration-dependent manner (Figure 2). The doses inducing 50% cell growth inhibition (IC 50 ) against K562 and HL-60 cells for CH 2 Cl 2 and EtOAc fractions extract of S. luteocoerulea are presented in Table 1. We used 700 nM of Paclitaxel as a positive control.  Figure 2. Dose-dependent growth inhibition of K562 and HL-60 cells by solvent fractions of S. luteo-coerulea (0-500 µg/mL) after 48 h. Viability was quantitated by MTS assay. The dose inducing IC 50 against K562 and HL-60 by CH 2 Cl 2 , EtOAc, solvent fractions of S. luteo-coerulea were calculated 94.39, 1093, 53.96, and 528.8 respectively. Paclitaxel (700 nM) was used as a positive control. Results are expressed as mean±SEM (n = 3). * p<0.05, * * p<0.01, and * * * p<0.001 compared to control.

Apoptosis induction by CH 2 Cl 2 fraction in K562 and HL-60 cells
Cells treated with CH 2 Cl 2 solvent fraction of S. luteo-coerulea were stained with PI and the percentage of apoptotic cells following treatment was measured by flow cytometry aiming to detect the sub-G1 peak resulting from DNA fragmentation. The K562 and HL-60 cells treated with 15, 30, 60, 125, and 250 µg/mL CH 2 Cl 2 solvent fraction of S. luteocoerulea for 48 h induced a sub-G1 peak in flow cytometry histogram compared to untreated control cells (Figure 3). Figure 3. Flow cytometry histograms of apoptosis assays by PI method in K562 and HL-60 cells. Cells were treated with different concentration of CH 2 Cl 2 solvent fractions of S. luteo-coerulea (0, 15, 30, 60, and 125 µg/mL) for 48 h. Sub-G1 peak as an indicative of apoptotic cells, was induced in CH 2 Cl 2 solvent fractions of S. luteo-coerulea treated but not in control cells. CH 2 Cl 2 fraction-treated cells exhibited a sub-G1 peak in K562 and HL-60 cells in a concentration-dependent manner that indicates the involvement of an apoptotic process in CH 2 Cl 2 fraction-induced cell death.

Discussion
The anti-proliferative and cytotoxic effect of many Scutellaria species on several cancer cell lines have been reported previously (Salini et al., 2013;Tayarani-Najaran et al., 2010;Tayarani-Najarani et al., 2012). In the present study, the cytotoxic properties of fractions of S. luteo-coerulea were investigated on K562 and HL-60. Fractions were tested for cytotoxic activity for range of 0-500 μg/mL after 48 h of treatment.
Wang et al., reported potent cytotoxic activities of eight crude extracts of five Scutellaria species against brine shrimps and four human cancer cell lines (HCA, HepG2, MCF-7, HPC) (Wang et al., 2010). In another study, in vitro antiproliferative screening investigation of crude methanol extracts of S. dominica L. leaves, S. lanigera Desf. aerial parts, S. menthaefolia Ten. roots, S. palaestina Benth. aerial parts, S. sclarea L. roots, and S. spinosa L. aerial parts have shown cytotoxic effects with IC 50 values ranged from 90 to 400 μg/mL (Sonoda et al., 2004). Different researches have been performed on active component of the species including baicalin, baicalein, and neobaicalein. Baicalein inhibited the proliferation of estrogen receptor positive human breast cancer MCF-7 cells in vitro (Wang et al., 2010). Baicalin and neobaicalein were found to induce apoptosis in human promyelocytic leukemia, HL-60, and HeLa cells, respectively through multiple pathways and may be an interesting strategy in treatment of leukemia. Pro-apoptotic mechanism of baicalin in leukemia Jurkat cells has been verified with characteristic morphologic change of apoptosis (Lamer-Zarawska et al., 2010). Baicalin also presented antiviral activity against viruses which have been identified as carcinogens. The anti-inflammatory effect of the Scutellaria flavones via downregulation of several inflammationassociated genes in malignant tumors is known to exert both pro-and antitumor effects (Lamer-Zarawska et al., 2010).
In our study, fractions obtained from S. luteo-coerulea had cytotoxic and apoptotic activities on K562 and HL-60 cells. In order to gain insight into the nature of the active principles responsible for the cytotoxic activity, the methanol extract was fractionated using solvents of increasing polarity. The IC 50 values of the fractions are compared in Table  1. Active constituent(s) of intermediate polarity is thus likely to be responsible for the observed cytotoxicity and future bioassay guided fractionation needs to focus on the CH 2 Cl 2 fraction. These results suggest that CH 2 Cl 2 fraction obtained from S. luteo-coerulea could be used as a potential apoptosis inducing agent, and that the CH 2 Cl 2 fraction obtained from S. luteocoerulea consists of a key component for cytotoxic activity. Regarding the sequential extraction with solvents of ascending polarity, the fraction of S. luteo-coerulea with CH 2 Cl 2 generated fractions that were cytotoxic for tumor cells. This can be explained by the low polarity of the solvents used in these fractions, which extract low-polar compounds that are either successfully absorbed through the cell or have cytotoxic activity. Therefore, it is presumed that most of the cytotoxic compounds or compounds with the highest potency were concentrated in the CH 2 Cl 2 fraction.
In conclusion, this study determined an anti-cancer effect of CH 2 Cl 2 fraction obtained from S. luteo-coerulea mediated by the induction of apoptosis, which is associated with DNA fragmentation in K562 and HL-60 cells.
Due to potential value of apoptosis in cancer therapeutic strategies, these results confirm the possible worth of CH 2 Cl 2 fraction obtained from S. luteo-coerulea as cytotoxic agents. CH 2 Cl 2 fraction can extract a compound that has considerable cytotoxic activity. However, further investigations on the active principals of CH 2 Cl 2 fraction obtained from S. luteo-coerulea are necessary to be elaborated.